The present invention relates to the identification of receptors for and functions of GDNF, and cell lines expressing the receptors.
Glial cell line-derived neurotrophic factor (GDNF) is a trophic polypeptide. It is a disulfide bridge-linked homodimer of two 134-amino acids long glycosylated polypeptides, with a molecular weight of approximately 25-30 kD for each monomer. Prior to the molecular cloning of GDNF in 1993, investigators sought a trophic polypeptide which would alleviate the neuronal loss associated with Parkinson""s disease, specifically dopaminergic neurons of the ventral mesencephalon. The survival of this subpopulation of neurons has been known for some time to be promoted by soluble factors present in the conditioned media of glial cell lines. It was from one of these cell lines that the GDNF protein was initially isolated based upon its ability to promote dopamine uptake in primary cultures prepared from embryonic ventral midbrain neurons (Lin et al., 260 Science 1120, 1993). Subsequently, GDNF was shown to promote survival of adult substantia nigra neurons in vivo following pharmacological treatments and lesions that mimic Parkinsonian syndromes (Beck et al., 377 Nature 339, 1995; Tomac et al., 373 Nature 335, 1995) Although GDNF was originally reported to be highly specific for dopaminergic neurons, several other potent activities of this molecule have subsequently been demonstrated, including survival and phenotypic responses in facial and spinal motor neurons (Henderson et al., 266 Science 30 1062, 1994; Oppenheim et al., 373 Nature 344, 1995; Yan et al., 373 Nature 341, 1995), noradrenergic neurons of the locus coeruleus (Arenas et al., Neuron, in press, 1995), cerebellar Purkinjie cells (Mount et al., 92 PNAS 9092, 1995), sympathetic and sensory neurons in peripheral ganglia (Trupp et al., 130 J. Cell Biol. 137, 1995) and for populations of peripheral neurons with target-derived and paracrine mode of action (Trapp, M. et. al., J. Cell Biol., 130, 137-148 (1995); Pitchel, J., Sariola, H., Hoffer, B. and Westphal, H. (unpublished observation); Buj-Bello, A., Buchman, V. L., Horton, A., Rosenthal, A.and Davies, A. M. Neuron, 15, 821-828 (1995). As many of these neurons are affected in neurodegenerative diseases, GDNF may have potent therapeutical applications. Particularly, exogenously administered GDNF maintains dopaminergic neurons of the substantia nigra in experimentally induced Parkinsons disease in rodents (Beck et al. (1995) Nature, 373, 339-341; Tomac et al. (1995) Nature, 373, 335-339) and leads to functional recovery in Parkinsonian rhesus monkeys (Gash et al. (1996) Nature, 380, 252-255). GDNF treatment also rescues about half of the experimentally axotomized murine motoneurons (Oppenheim et al. (1995) Nature, 373, 344-346; Li et al. (1995) Proc. Natl, Acad. Sci. U.S.A., 92, 9771-9775) suggesting that GDNF may be used in treatment of motoneuronal diseases. The studies of the mechanism of GDNF action in normal and pathogenic conditions have been, however, basically hampered as its receptor was not known.
Based upon structural similarities (primarily seven conserved cysteine amino acid residues), GDNF appears to be a distant member of the transforming growth factor-beta (TGF-13) superfamily of multifunctional cytokines, which includes TGF-xcex2s, activins, bone-morphogenetic proteins (BMPs) and growth and differentiation factors (GDFS) (Roberts et al., 327 Philos.Trans.R.Soc.Land. 145,1990). TGF-xcex2 and related ligands are known to suppress proliferation in epithelial and immune cells, to function as morphogens in early development, to induce ectopic expression of skeletal tissue, and to promote survival and differentiation of neurons. TGF-xcex2 superfamily proteins interact with numerous receptor subunits on the surface of responsive cells (Attisano et al., 1222 Mol. Cell Res. 71, 1994; Derynck, 19 Trends Biochem. Sci. 548, 1994). Different receptor types have been described based on the molecular weights of affinity labeled complexes. Among these are the type I, type II and type III receptors, which represent binding proteins of 55 kD, 70 kD and 300 kD, respectively. Type III receptors are abundantly expressed transmembrane proteoglycans of approximately 300 kD with a short cytoplasmic tail, and are thought to function in recruitment of ligand to an oligomeric receptor complex (Lopez-Casillas et al., 67 Cell 785, 1991). Indeed, a type III receptor is required on some cell lines for TGF-xcex22 binding to the signaling receptors. Type I and type II receptors are transmembrane proteins with an intracellular serine-threonine kinase domain and can therefore transmit downstream signals upon ligand binding (Attisano et al., 75 Cell 671, 1993; Derynck, 1994 supra). Type II receptors are constitutively activated kinases which upon ligand binding recruit type I receptors to a signaling complex. In this complex, type I receptors are phosphorylated by type II receptors on a juxtamembrane domain rich in serine residues, this phosphorylation is thought to result in the activation of the ser-thr kinase activity of type I receptors and in downstream signaling (Wrana et al., 370 Nature 341, 1994). According to this model, TGF-xcex2 superfamily proteins can not bind to type I receptors in the absence of type II receptors, although in some cases, type I receptors are necessary for efficient binding to type II receptors (Letsou et al., 80 Cell 899, 1995). Multiple cDNA clones of type I, II and III receptors for TGF-xcex2S, activins and BMPs have been isolated by either expression or homology cloning, including seven mammalian type I receptors, four type II receptors and one type III betaglycan receptor. Additional membrane proteins binding different members of this family include glycosylphosphatidyl inositol (GPS)-linked 150 kD and 180 kD proteins of unknown structure and function (MacKay and Danielpour, 266 J. Biol. Chem. 9907, 1991), and endoglin, a 180 kD disulphide linked dimer which binds TGF-xcex21 but not TGF-xcex22.
The isolation and characterization of GDNF receptors is a prerequisite for the understanding of the full range of biological actions of GDNF and the signaling events that take place upon GDNF binding to responsive cells. Until now, progress in this area has been hampered by the lack of cell lines responsive to GDNF, that is, cell lines comprising GDNF receptors.
Receptors for GDNF are disclosed herein, as are cell lines expressing the same. Methods for identifying and isolating these receptors are also disclosed.
In one aspect, the present invention relates to isolated receptors which bind GDNF.
In another aspect, the present invention relates a method for determining compounds or compositions which bind GDNF receptors.
In yet another aspect, the present invention relates to methods for identifying homologs of GDNF by screening for compounds or compositions which have similar biological effects, such as tyrosine phosphorylation, increase in c-fos mRNA, and increases in cell survival.
In still another aspect, the present invention relates to methods for identifying analogs of GDNF by screening for compounds or compositions which are antagonistic for the biological effects of GDNF, such as are listed above.
In a further aspect, the present invention relates to compounds having the sequence as set forth in SEQ ID NOS:2 and 9.
In yet a further aspect, the present invention relates to nucleic acids having the sequence as set forth in SEQ ID NOS:5 and 10.